Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection
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viruses Article Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection Ting-Chun Hung 1,2, Alagie Jassey 3, Chien-Ju Lin 4, Ching-Hsuan Liu 5,6, Chun-Ching Lin 1,4, Ming-Hong Yen 1,4,* and Liang-Tzung Lin 5,7,* 1 Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 807 Kaohsiung, Taiwan; [email protected] (T.-C.H.); [email protected] (C.-C.L.) 2 Department of Clinical Pathology, Chi Mei Medical Center, Tainan 710, Taiwan 3 International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] 4 School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan; [email protected] 5 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] 6 Department of Microbiology & Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada 7 Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan * Correspondence: [email protected] (M.-H.Y.); [email protected] (L.-T.L.); Tel: +886-7-312-1101 ext. 2665 (M.-H.Y.); +886-2-2736-1661 ext. 3911 (L.-T.L.) Received: 18 September 2018; Accepted: 25 November 2018; Published: 27 November 2018 Abstract: Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identified RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent. Keywords: HCV; Rhizoma coptidis; herbal medicine; antiviral; entry inhibition 1. Introduction Hepatitis C virus (HCV) is an important liver pathogen belonging to the Flaviviridae family with an enveloped positive single-stranded RNA genome. HCV has seven genotypes (genotype 1~7) and a genome size of about 9.6 kb which encodes a polyprotein that is approximately 3000 amino acids long. The polyprotein upon translation is processed by viral and host proteases to yield 10 matured protein including structural proteins, Core, E1, E2, and p7 ion channel, as well as non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B [1]. HCV entry into the host hepatocytes is mediated by interaction Viruses 2018, 10, 669; doi:10.3390/v10120669 www.mdpi.com/journal/viruses Viruses 2018, 10, 669 2 of 12 with several notable cell surface and tight junction receptors/co-receptors including heparin sulfate proteoglycans (HSPG), cluster of differentiation 81 (CD81), low density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1), and occludin (OCLN) [2,3]. Additional factors that can influence viral entry include apolipoprotein E (ApoE), which is incorporated on infectious HCV virions [4], and can function as an exchangeable apolipoprotein between secreted ApoE-associated lipoproteins and the HCV lipoviroparticle (LVP) to enhanced HCV infection [5]. There are over 170 million HCV carriers worldwide. HCV infection can lead to chronic hepatitis, cirrhosis, and liver cancer, and there is still no effective vaccine against the virus. While the previous standard of care consisting of PEGylated-interferon (IFN)-α in combination with ribavirin is associated with several important drawbacks including severe side effects and low efficacy against HCV genotype 1, the recent introduction of the direct-acting antivirals (DAAs) targeting the viral non-structural proteins has substantially improved the sustained virological response (SVR) in the most difficult to treat genotype 1 patients [6]. However, the DAAs also have challenges including potential toxicity, especially from drug-drug interactions (DDIs). For instance, HCV protease inhibitors are at high risk for DDIs as they are known substrates and inhibitors of cytochrome P450 (CYP) 3A4 system and can interfere with the metabolism of other drugs including immunosuppressants (e.g. cyclosporine and tacrolimus) when co-administered in liver transplant setting [7]. Other drug-drug interactions from HCV DAAs include those with acid-suppression therapies (e.g. famotidine and omeprazole) or the human immunodeficiency virus (HIV) antiretroviral agents (e.g. Rilpivirine and Efavirenz), which have been shown to decrease the effectiveness of the HCV NS5A inhibitor Ledipasvir [8] and produce adverse drug reactions with the protease inhibitor Paritaprevir [7], respectively. In addition, due to the great genetic variability of HCV, selection of resistant mutants is becoming a challenge as a greater number of people are being treated in real-world settings, which can potentially lead to DAA failures [6,9]. Therefore, continuous identification of novel candidate drugs particularly with a different mode of action to improve the current therapeutic strategies is highly envisaged. Rhizoma Coptidis (RC) is the dried rhizome typically obtained from Coptis chinensis Franch (‘Chinese goldthread’), which is a medicinal plant of the Ranunculaceae family [10]. RC is one of the most commonly used Chinese medicinal herbs (also known as ‘Huang Lian’) documented by the WHO [10] and is known to contain various bioactive alkaloids [11]. It is traditionally used for its “heat clearing” and “detoxification” effects such as treatment against arthritis, burns, eczema, microbial infections, and gastrointestinal diseases [10,12,13]. In addition, RC’s traditional usage against infections has been correlated through several recent studies that validated its antimicrobial functions, including against several bacteria and viruses [13]. Specifically, RC and its major constituents have been shown to exert inhibitory effects against herpesvirus, respiratory syncytial virus, and mouse hepatitis A virus infections [14–16]. These precedents suggest that RC may be a potent source for the discovery of novel antiviral treatments. Since the effect of RC on HCV infection remains largely unexplored, and in an attempt to identify novel anti-HCV agents, in this study we examined the impact of the methanolic extract of RC on HCV infection. Our results demonstrated that RC could robustly inhibit HCV infection by targeting the early steps in viral entry. Specifically, the targeted steps included viral attachment and entry/fusion into the host cells. In addition, the RC-mediated inhibition of HCV is not genotype-specific as the drug equally inhibits other HCV genotypes, thereby identifying RC as a potential pan-genotypic anti-HCV agent. 2. Materials and Methods 2.1. Cell Culture and Virus Production Culture of Huh-7.5 cells (human hepatoma, Huh-7 cell derivative) and production of cell-culture derived HCV particles (HCVcc) from the Gaussia luciferase reporter-tagged Jc1FLAG2(p7-nsGluc2A) construct (genotype 2a; kindly provided by Dr. Charles M. Rice) were carried out as previously described [17]. Virus concentration was expressed as multiplicity of infection (MOI) and the Viruses 2018, 10, 669 3 of 12 basal media for all viral infection analyses consisted of Dulbecco’s Modified Eagle’s Medium (GIBCO-Invitrogen, Carlsbad, CA, USA) containing 2% fetal bovine serum. 2.2. Plant Extract Preparation Rhizoma Coptidis roots from Coptis chinensis Franch (ID#kew-2736105 from The Plant List [18]) were obtained from local pharmacy store (Kaohsiung, Taiwan) and authenticated by Dr. Ming-Hong Yen using anatomical methods as well as by HPLC analysis through comparison to known molecular standards as previously described [10,19]. A voucher specimen was deposited at the Kaohsiung Medical University herbarium (CTM-RCC03). For methanol extraction [20], the roots were washed, dried, and homogenized before extraction with 100 % methanol, followed by concentration in vacuo. The methanolic RC stock was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) prior to use. 2.3. Cytotoxicity Assay and Antiviral Activity Analysis Huh-7.5 cells seeded at 1 × 104 cells/well in 96-well plates overnight were treated with increasing concentrations of RC for 5 days. The cells were then washed twice with phosphate buffered saline (PBS) before XTT cell viability analysis as previously described [21]. For examining antiviral activity, Huh-7.5 cells (1 × 104 cells/well in 96-well plates) were concurrently treated with the virus (MOI = 0.01) and the test drug at various concentrations before